Chen_2018_J.Mol.Graph.Model_85_182

The esterase RhEst1 can catalyze the asymmetric hydrolysis of ethyl (+/-)-2,2-dimethylcyclopropane carboxylate (DmCpCe), yielding a pharmaceutically relevant (S)-carboxylic acid. A triple mutant RhEst1A147I/V148F/G254A showed a 5-fold increase in the catalytic activity but a significant decrease in the enantioselectivity. Further optimization studies led to a new enzyme with an additional A143T mutation, which showed both increased catalytic activity and recovered enantioselectivity as well as improved thermostability. To reveal the detailed structural mechanisms for these improved properties, we performed all-atom molecular dynamics simulations on the wild type and two mutants A147I/V148F/G254A and A143T/A147I/V148F/G254A RhEst1, in complex with R-DmCpCe and S-DmCpCe substrates, respectively. The structural stability of the enzyme variants was investigated with the residue interaction network analysis. In RhEst1M2, S-DmCpCe was observed to adopt a more "activated" conformation than R-DmCpCe, with the active site residues better prearranged for the reaction, leading to the improved enantioselectivity towards S-DmCpCe. The mutations in the two mutants, especially A143T, could lead to different motion patterns in the cap domain, thus affecting the structure of the substrate entrance tunnel. The residue interaction networks analysis showed an increased number of interactions in RhEst1M1 and RhEst1M2 as compared to the wild type enzyme, especially the pi-pi stacking interactions between Phe148 and the nearby residues, providing an explanation for the increased thermostability of the two mutant enzymes observed experimentally. Our work provides essential molecular insights into the substrate binding, enantioselectivity and structural stability of esterase RhEst1, which will facilitate the development of more efficient RhEst1 variants for pharmaceutical applications.